The present invention relates to recombinant production of carotenoids and biological materials useful therefor.
Phaffia rhodozyma (P. rhodozyma) is a carotenogenic yeast strain which produces astaxanthin. Astaxanthin is distributed in a wide variety of organisms such as animals (birds such as flamingo and scarlet ibis, and fish such as rainbow trout and salmon), algae and microorganisms. It is also recognized that astaxanthin has a strong antioxidation property against oxygen radicals, and is expected to be useful pharmaceutically for protecting living cells against certain diseases, such as a cancer. Moreover, industrial need for astaxanthin as a coloring reagent is increasing, especially in the industry of farmed fish like salmon, because astaxanthin imparts a distinctive orange-red coloration to the animals and contributes to consumer appeal in the marketplace.
P. rhodozyma is known as a carotenogenic yeast strain which produces astaxanthin. Different from the other carotenogenic yeast, Rhodotorula species, P. rhodozyma can ferment some sugars such as D-glucose. This is an important feature from a viewpoint of industrial application. In a recent taxonomic study, a sexual cycle of P. rhodozyma was revealed and its telemorphic state was designated under the name of Xanthophyllomyces dendrorhous (W. I. Golubev; Yeast 11, 101-110, 1995). Some strain improvement studies to obtain hyper producers of astaxanthin from P. rhodozyma have been conducted, but such efforts have been restricted to employ the method of conventional mutagenesis and protoplast fusion in this decade. Recently, Wery et al. developed a host vector system using P. rhodozyma in which a non-replicable plasmid was integrated in multiple copies into the genome of the ribosomal DNA of P. rhodozyma (Wery et al., Gene, 184, 89-97, 1997). Verdoes et al. reported more improved vectors to obtain a transformant of P. rhodozyma as well as its three carotenogenic genes which code for the enzymes that catalyze the reactions from geranylgeranyl pyrophosphate to beta-carotene (WO 97/23633).
A specific biosynthetic pathway for carotenogenesis branches from the general isoprenoid pathway at the point of an important intermediate, farnesyl pyrophosphate (FPP) (FIG. 1). FPP and IPP are condensed by geranylgeranyl pyrophosphate (GGPP) synthase which is encoded by crtE in P. rhodozyma to produce GGPP. GGPP is then converted to beta-carotene by the sequential reaction of an enzyme functioning doubly as phytoene synthase and lycopene cyclase which is encoded by crtBY and phytoene desaturase encoded by crtI.
In bacteria, enzymes and genes which are involved in xanthophyll formation have been isolated and characterized in detail. Beta-carotene hydroxylase which is coded by crtZ is involved in the two steps of hydroxylation for the beta-ionone-ring of beta-carotene at both of the ends. The crtZ gene has been cloned from a wide variety of organisms such as Erwinia uredovora (Misawa et al., J. Bacteriol., 172, 6704-6712, 1990), Flavobactor species (L. Pasamontes et al., 185 (1), 35-41, 1997) and Agrobacterium aurantiacum (Misawa et al., J. Bacteriol., 177 (22), 6575-6584, 1995). Beta-carotene ketolase which is encoded by crtW catalyzes the two steps of introduction of an oxo-group into the beta-ionone -ring of beta-carotene at both of the ends. Kajiwara et al. cloned and sequenced the bkt gene corresponding to crtW in eubacteria from Haematococcus bluvialis (Kajiwara et al., P. Mol. Biol., 29, 343-352, 1995). Harker et al. also cloned and sequenced the crtO gene corresponding to crtW in eubacteria from Synechococcus PCC7942 (Harker et al., FEBS Letters, 404, 129-134, 1997). Both enzymes, i.e., the hydroxylase and the ketolase, have wide substrate specificity and this ensures the formation of a wide variety of xanthophylls in case both of the enzymes react at the same time, depending on the reaction condition. (FIG. 1)
As described above, all the genes which were involved in the formation of beta-carotene from FPP have been isolated but the enzymes and genes which would be involved in the last step of xanthophyll formation from beta-carotene have not been identified on the protein and DNA level in P.rhodozyma. Although Johnson et al. (Crit. Rev. Biotechnol, 11 (4), 297-326, 191) proposed the existence of two independent pathways for astaxanthin formation by assuming that some of the xanthophyll compounds isolated by them would be intermediates of astaxanthin biosynthesis, these two independent pathways could not be proven because enzymes and genes which are involved in such pathways could not be isolated. Furthermore, it can not be excluded that these xanthophyll compounds could have resulted from an experimental artifact in the isolation step of these compounds. Failure to isolate a mutant from P. rhodozyma which accumulates intermediates in the biosynthetic pathway from beta-carotene to astaxanthin made it difficult to clarify the biosynthetic pathway from beta-carotene to astaxanthin.
This invention relates to a gene and an enzyme which is involved in the last step of astaxanthin biosynthesis (i.e., from beta-carotene to astaxanthin).
The present invention provides an isolated DNA, for example, a cDNA including a nucleotide sequence coding for astaxanthin synthase which is involved in the reaction from beta-carotene to astaxanthin in P. rhodozyma, like the AST gene.
In a preferred embodiment, the cloned DNA fragment can be characterized in that
(a) the nucleotide sequence encodes an enzyme having the amino acid sequence described in SEQ ID NO: 1, or
(b) the nucleotide sequence encodes a variant of the enzyme selected in (a), which nucleotide sequence is either (i) an allelic variant or (ii) an enzyme having one or more amino acid insertions, deletions, and/or substitutions and having the stated enzyme activity.
In another preferred embodiment, the isolated cDNA fragment can be derived from a gene of Phaffia rhodozyma and is selected from:
(i) a cDNA sequence represented by SEQ ID NO: 2;
(ii) an isocoding or an allelic variant for the cDNA sequence represented by SEQ ID NO: 2; and
(iii) a derivative of a cDNA sequence represented by SEQ ID NO: 2 with insertions, deletions, and/or substitutions of one or more nucleotide(s), and encoding a polypeptide having the enzyme activity.
In another preferred embodiment, the present invention includes the isolated cDNA as described above, which is characterized in that the nucleotide sequence is:
(i) a nucleotide sequence represented in SEQ ID NO: 2;
(ii) a nucleotide sequence which, because of the degeneracy of the genetic code, encodes an astaxanthin synthase having the same amino acid sequence as that encoded by the nucleotide sequence in (i); and
(iii) a nucleotide sequence which hybridizes to the complement of the nucleotide sequence from i) or ii) under standard hybridizing conditions.
In still another preferred embodiment, an isolated genomic DNA fragment can be derived from a gene of Phaffia rhodozyma and is selected from:
(i) a genomic DNA sequence represented by SEQ ID NO: 3;
(ii) an isocoding or an allelic variant for the genomic DNA sequence represented by SEQ ID NO: 3; and
(iii) a derivative of a genomic DNA sequence represented by SEQ ID NO: 3 with insertions, deletions, and/or substitutions of one or more nucleotide(s), and coding for a polypeptide having the enzyme activity.
In another preferred embodiment the present invention includes the isolated genomic DNA as described above, which is characterized in that the nucleotide sequence is:
(i) a nucleotide sequence represented in SEQ ID NO: 3;
(ii) a nucleotide sequence which, because of the degeneracy of the genetic code, encodes an astaxanthin synthase having the same amino acid sequence as that encoded by the nucleotide sequence in (i); and
(iii) a nucleotide sequence which hybridizes to the complement of the nucleotide sequence from i) or ii) under standard hybridizing conditions.
Another aspect of the present invention is a recombinant polypeptide having astaxanthin synthase activity and which is involved in the reaction from beta-carotene to astaxanthin in P. rhodozyma which is obtainable by the expression of the cloned DNA fragment as set forth above.
A preferred embodiment of the recombinant polypeptide of the present invention is characterized in that
(a) the polypeptide has an amino acid sequence as described in SEQ ID NO: 1, or
(b) the polypeptide is a variant of the peptide defined in (a) which is selected from (i) an allelic variant or (ii) an enzyme having one or more amino acid insertions, deletions and/or substitutions and having the stated enzyme activity.
The present invention also includes variants of the polypeptides set forth above. Such variants are defined on the basis of the amino acid sequence of the present invention by insertions, deletions, and/or substitutions of one or more amino acid residues of such sequences wherein such variants still have the same type of enzymatic activity as the corresponding polypeptides of the present invention or they are the result of the well known phenomenon of allelic variation. Such activities can be measured by any assays known in the art or specifically described herein. Such variants can be made either by chemical peptide synthesis known in the art or by recombinant means on the basis of the DNA sequences as disclosed herein by methods known in the state of the art, such as, e.g. that disclosed by Sambrook et al. (Molecular Cloning, Cold Spring Harbour Laboratory Press, New York, USA, second edition 1989).
Amino acid exchanges in proteins and peptides which do not generally alter the activity of such molecules are known in the state of the art and are described, for example, by H. Neurath and R. L . Hill in xe2x80x9cThe Proteinsxe2x80x9d (Academic Press, New York, 1979, see especially FIG. 6, page 14). The most commonly occurring exchanges are: Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro, Lys/Art, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly as well as the reverse. It is also possible to add or delete one or several amino acid residues(s) at N- and/or C-terminal of the enzyme without any critical effect on the activity of the present synthase.
Furthermore, the present invention is not only directed to the DNA sequences as disclosed e.g., in the sequence listing as well as their complementary strands, but also to those which include these sequences, DNA sequences which hybridize under standard conditions with such sequences or fragments thereof and DNA sequences, which because of the degeneration of the genetic code, do not hybridize under standard conditions with such sequences but which code for polypeptides having exactly the same amino acid sequence.
The said enzyme activity is expressed as the enzyme activity which renders astaxanthin production to beta-carotene producing microorganism by means of the transformation to express its corresponding gene in the said beta-carotene producing host organisms.
The said enzyme activity is also expressed as the enzyme activity which renders astaxanthin production to microorganism which accumulates intermediate xanthophyll from beta-carotene to astaxanthin described in FIG. 1; e.g. echinenone, beta-cryptoxanthin, canthaxanthin, 3xe2x80x2-hydroxyechinenone, 3-hydroxyechinenone, zeaxanthin, phoenicoxanthin and adonixanthin.
The said enzyme activity can also be expressed as the enzyme activity which catalyzes the astaxanthin formation from various substrates such as beta-carotene, echinenone, beta-cryptoxanthin, canthaxanthin, 3xe2x80x2-hydroxyechinenone, 3-hydroxyechinenone, zeaxanthin, phoenicoxanthin and adonixanthin under appropriate in vitro condition which constitutes of membrane fraction such as natural membrane like microsome and artificial membrane like liposome in company with appropriate electron donor like NADPH.xe2x80x9d
In the present invention, unless otherwise indicated, the hybridization reactions are generally carried out at 42xc2x0 C., which is 15 to 35xc2x0 C. below the Tm of most DNA probes, thus ensuring a maximum rate of hybridization. The desired stringency of hybridization is achieved by washing, e.g., the filter at a salt concentration and temperature that is approximately 5 to 15xc2x0 C. below the Tm for a perfectly matched hybrid. The salt concentration and temperature, however, may be adjusted to less stringent conditions if significant mismatching of sequence is expected (e.g., when probing for the same gene in a different species or for a different but related sequence).
As used herein, the phrase xe2x80x9cstandard conditionsxe2x80x9d for hybridization means the conditions which are generally used by a person skilled in the art to detect specific hybridization signals and which are described, e.g. by Sambrook et al., (s.a.) or preferably so-called stringent hybridization and non-stringent washing conditions, or more preferably so-called stringent hybridization and stringent washing conditions a person skilled in the art is familiar with and which are described, e.g., in Sambrook et al. (s.a) or more preferably so-called medium stringent conditions, e.g. using the DIG (digoxigenin) labeling kit and luminescent detection kit of Boehringer Mannheim (Mannheim, Germany) following the protocol given by the manufacturer and using as the hybridization solution:
formamide (WAKO, Osaka, Japan) 50% (V/V)
5xc3x97SSC
blocking reagent (Boehringer) 2% (W/V)
N-lauroylsarcosine 0.1% (W/V)
SDS 0.3% (W/V)
at a temperature of 42xc2x0 C. over night and subsequently washing and detection as indicated by the manufacturer.
For example, a typical wash sequence includes washing the hybridized blot first with a solution A containing 2xc3x97SSC/0.1% SDS in water at room temperature. Next, the blot is washed twice in solution B containing 0.1xc3x97SSC/0.1% SDS in water at a temperature to be determined based on the desired level of stringency. For example, a perfectly matched hybrid may be washed at a temperature from about 55xc2x0 to about 65xc2x0 C.; for a probe from a related gene or from a different species, the wash temperature may be, for example, from about 37xc2x0 C. to about 52xc2x0 C. Unless otherwise indicated, this washing condition was used in the present invention.
DNA sequences which are derived from the DNA sequences of the present invention either because they hybridize with such DNA sequences (see above) or can be constructed by the polymerase chain reaction by using primers designed on the basis of such DNA sequences can be prepared either as indicated, namely by the PCR reaction, or by site directed mutagenesis (see e.g., Smith, Ann. Rev. Genet. 19, 423 (1985)) or synthetically as described, e.g., in EP 747 483 or by the usual methods of Molecular Cloning as described, e.g., in Sambrook et al. (s.a.).
The present invention also includes a vector or plasmid that contains a DNA as described above and a host cell transformed or transfected by a DNA as described above or a vector or plasmid as indicated above.
The present invention also provides a recombinant organism which is obtainable by the transformation of a host using a recombinant DNA carrying the DNA as mentioned above.
The present invention also includes a method for producing an enzymatic polypeptide capable of catalyzing the reaction from beta-carotene to astaxanthin, which includes culturing a recombinant organism described above under conditions conductive for the production of the enzymatic polypeptide.
In a further aspect, the present invention provides a method for the production of astaxanthin which includes introducing one or more of the DNAs described above into an appropriate host organism and cultivating this transformed organism under conditions conductive for the production of astaxanthin.
The enzymatic polypeptide of the present invention is also useful in a method for producing astaxanthin, which method includes contacting beta-carotene with a recombinant polypeptide having an astaxanthin synthase activity involved in the reaction from beta-carotene to astaxanthin as set forth above in the presence of an appropriate electron donor in an appropriate reaction mixture containing an appropriate reconstituted membrane. In this method, the recombinant polypeptide may be present in the form of a reconstituted membrane which is prepared from biological membranes such as, for example, microsomes or mitochondrial membranes. The recombinant polypeptide may be also present in the form of a reconstituted artificial membrane, such as for example, a liposome. An electron donor, such as, cytochrome P450 reductase is an example of an appropriate electron donor which can reduce a reaction center of the enzyme of the present invention.
Another embodiment of the invention is an isolated polynucleotide encoding a polypeptide which is SEQ ID NO:1, an isolated polynucleotide which is SEQ ID NO:2, or an isolated polynucleotide which is SEQ ID NO:3.
Another embodiment of the invention is a polypeptide having astaxanthin synthase activity which is SEQ ID NO:1. The present invention also includes a vector containing a polynucleotide which encodes SEQ ID NO:1, a polynucleotide which is SEQ ID NO:2, or a polynucleotide which is SEQ ID NO:3. A host cell is also provided which is transformed with the vector set forth above.
In another embodiment, the present invention provides a process for producing astaxanthin which includes: (a) cultivating in a suitable culture medium a recombinantly produced host cell containing a polynucleotide which encodes a polypeptide having astaxanthin synthase activity.